There is evidence that high density lipoprotein particles within the ovarian follicle play a role in female reproductive potential. Follicular fluid (FF) is a biological window which can reveal the metabolic processes occurring in the microenvironment of the maturing oocyte. Identification of HDL biomarkers in FF that predict early embryo development outcome parameters would enable a greater understanding of the factors that regulate oocyte competence during in vitro fertilization (IVF). This gain of knowledge would allow for further research development in the understanding of the genetic and environmental regulation of oocyte development within the human follicle. High density lipoprotein (HDL) is the sole lipoprotein present in FF. The cholesterol transport, anti-oxidant and anti-inflammatory activities of HDL are a function of the dynamic HDL particle size distributions and the complex HDL composition which includes lipids, apolipoproteins, micronutrients and enzymes. In pilot studies, we have established that HDL cholesterol and apolipoprotein AI (Apo-AI) concentrations, reflecting total HDL concentration within FF, are significant negative predictors of detrimental embryo fragmentation during IVF. Furthermore, we have established that serum and FF HDL phospholipid: cholesterol molar ratios (reflecting HDL particle size and subclass distribution) are positively correlated with embryo fragmentation. We have also directly demonstrated by NMR spectrometry and HDL subclass isolation and analysis that FF-HDL particles have an altered size distribution and lipid composition relative to serum. The presence of HDL-associated human serum paraoxonase 1 (PON1) activities in FF has also been originally described. A prospective cohort of 180 women undergoing IVF will be recruited. Follicular fluid, granulosa cells and peripheral blood will be collected during IVF treatment. Serum and FF-HDL particle size will be determined by NMR spectrometry and HDL subclass (HDL2 and HDL3) will be isolated and their associated lipid, apolipoprotein, fat-soluble micronutrient compositions, and PON1 activities will be assessed. All measurements will be correlated with embryo morphology parameters (surrogate markers of oocyte integrity). Normalization of HDL subclass components per HDL2 or HDL3 particle will be used to characterize the unique particle composition of FF-HDL relative to serum HDL and identify the specific differences which may account for our pilot observations with regard to embryo quality parameters. We will also examine the lipoprotein signaling and cholesterol metabolism-related gene expression from GCs obtained from embryos with/without fragmentation to identify differences in cholesterol metabolism genes. Aside from its importance as a window into the metabolic state of the human ovarian follicle, FF represents an excellent model system for studying HDL-mediated cholesterol metabolism and transport mechanisms in a physiologic system devoid of other lipoproteins. Understanding how modifications of HDL within FF render it dysfunctional would add greater depth of understanding to the role of HDL in the developmental potential of the human oocyte and embryo. PUBLIC HEALTH RELEVANCE: The work detailed in this study proposal focuses on better defining the physiology of the human ovarian follicle with respect to HDL particle metabolism that may influence the developmental potential of the human oocyte and embryo. Given the societal dilemma of reproductive choice with advanced maternal age resulting in the infertile condition together with the increasing evidence that environmental exposures influence various aspects of reproductive physiology, it is imperative to develop a greater understanding of the role that HDL plays in the health of the human ovarian follicle and the oocyte within.